The human leucocyte antigen (HLA), which represents major human histocompatibility complex (MHC), presents peptides derived from foreign proteins such as pathogens and peptides derived from self-proteins to T cells. In this manner, HLA is deeply involved in induction of immunological responses. As major HLAs, six types of antigens are known, namely, class I antigens (HLA-A, HLA-B, HLA-C), which is expressed in almost all cells, and class II antigens (HLA-DR, HLA-DQ, HLA-DP), which is expressed mainly in immune cells.
The HLA class I antigen consists of a highly polymorphic α chain and a substantially non-polymorphic β2-microglobulin; whereas the HLA class II antigen consists of a highly polymorphic β chain and a less polymorphic α chain. The α chains of class I antigens are encoded by HLA-A, HLA-B and HLA-C genes. The β chains of class II antigens are encoded by HLA-DRB1, HLA-DRB3/4/5, HLA-DQB1 and HLA-DPB1 genes, whereas the α chains are encoded by HLA-DRA1, HLA-DQA1 and HLA-DPA1 genes. In a gene level, in HLA class I antigens, exon 2 and exon 3 of a gene encoding an a chain are highly polymorphic; whereas, in HLA class II antigens, exon 2 of a gene encoding a β chain is highly polymorphic.
A gene region encoding a HLA is located on short arm of human chromosome 6 at 6p21.3. A Class I region (HLA-A, HLA-B and HLA-C, etc.), a class III region and a class II region (HLA-DRA, HLA-DRB1, HLA-DRB3/4/5, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, etc.) are arranged in this order from the telomere side toward the centromere side. Many genes are encoded at an extremely high density and association of these genes with transfusion, transplantation and various diseases have been reported. In the class III region, no HLA genes are present and genes of complement components and tumor necrosis factors (TNF), etc. are present.
In a HLA-DRB gene region encoding a β chain of a HLA-DR antigen, it has been confirmed that 5 types of structural polymorphisms are present. In DR1 type and DR10 type, pseudogenes such as HLA-DRB6 and HLA-DRB9 in addition to HLA-DRB1 are located on the same haplotype. In DR2 type, a HLA-DRB5 (DR51) gene and pseudogenes such as HLA-DRB6 and HLA-DRB9 in addition to HLA-DRB1 are located on the same haplotype. In DR3, DR5 and DR6 types, a HLA-DRB3 (DR52) gene and pseudogenes such as HLA-DRB2 and HLA-DRB9 in addition to HLA-DRB1 are located on the same haplotype. In DR4, DR7 and DR9 types, a HLA-DRB4 (DR53) gene and pseudogenes such as HLA-DRB7, HLA-DRB8 and HLA-DRB9 in addition to HLA-DRB1 are located on the same haplotype. In contrast to these, in DR8 type, no HLA-DRB genes except HLA-DRB1 are located on the same haplotype (FIG. 1).
In the exon of each HLA gene, regions having an abundance of polymorphism (polymorphic regions) are present. In many cases, a nucleotide sequence (amino acid sequence) present in a certain polymorphic region is commonly present in a plurality of HLA alleles. In short, each HLA allele is specified by a plurality of polymorphic regions in combination. In a HLA class I antigen, not only a polymorphic region in the exon but also exon 2 or exon 3 having the same nucleotide sequence is sometimes commonly present in a plurality of alleles.
Since a highly polymorphic region is present in a HLA, the number of types of HLA alleles is known to be extremely large and notation of them has been defined: i.e., a first field (two-digit level) is for discrimination of serologic HLA types, a second field (4-digit level) is for discrimination of alleles having an amino acid substitution in the same serologic HLA type, a third field (6-digit level) is for discrimination of alleles having a base substitution not accompanying an amino acid mutation and a fourth field (8-digit level) is for discrimination of alleles having a base substitution in an intron, which is out of the genetic region encoding a HLA molecule (FIG. 2).
In bone marrow transplantation, it is said that if the HLA type (HLA-A, HLA-B, HLA-C, HLA-DRB1) of a patient seeking to receive a transplant completely matches the HLA type of a donor at a 4-digit level, the success rate of transplantation improves and a severe graft versus host disease (GVHD) frequency reduces. Conversely, if the HLA types do not match at a 4 digit level, a risk of causing a failure such as a rejection response increases. Accordingly, accurate and highly precise HLA typing is extremely important also in a clinical point of view.
As a method for DNA typing in a HLA gene, a SBT (sequence based typing) method and a SSO (Sequence Specific Oligonucleotide)—Luminex method based on a polymerase chain reaction (PCR) are in mainstream.
These conventional DNA typing methods have an advantage in that typing of many samples is quickly performed; however, sometimes fail to accurately determine a polymorphic region and cis/trans positional relationship of exons on a chromosome in the case of a class I gene. Because of this, phase ambiguity occurs, highly precise HLA typing was sometimes not easily performed.
Since the conventional methods are DNA typing methods using PCR mainly based on exon regions of each HLA gene, base substitutions in an intron region and a promoter region are overlooked, with the result that there was a risk of failure in detection of a null allele, which has the same gene structure as other HLA expressing genes but is suppressed in expression.
For example, since the PCR conditions for each HLA gene are not same, the PCR for each HLA gene must be independently performed, thus, according to the method described in the Patent Document 1, an accelerated and simplified operation is not achieved.
The present inventors offered a highly precise DNA typing method capable of eliminating phase ambiguity by using a set of primers which respectively anneal specifically to an upstream region and a downstream region of each HLA gene locus (Patent Document 2 and Non Patent Document 2). However, since even in the method the PCR conditions for each gene locus are not completely unified, a multiple method in which PCR for all of the gene loci are simultaneously performed has never been achieved.
Furthermore, even though the acceleration of PCT is expected, a high-speed PCR apparatus in which the time required for the rise and fall of the temperature during the PCR is drastically reduced, has never been considered.